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    products/assay-kits/acetyl-coa-assay-kit-ab87546.pdf

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Acetyl CoA Assay Kit (ab87546)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (1)Q&A (18)References (48)

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Functional Studies - PicoProbe™ Acetyl CoA Assay Kit (ab87546)
  • Functional Studies - PicoProbe™ Acetyl CoA Assay Kit (ab87546)

Key features and details

  • Assay type: Quantitative
  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 20 min
  • Sample type: Cell Lysate, Tissue Lysate
  • Sensitivity: 0.01 nmol/well

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Overview

  • Product name

    Acetyl CoA Assay Kit
  • Detection method

    Fluorescent
  • Sample type

    Cell Lysate, Tissue Lysate
  • Assay type

    Quantitative
  • Sensitivity

    > 0.01 nmol/well
  • Range

    0.01 nmol/well - 1 nmol/well
  • Assay time

    0h 20m
  • Product overview

    Acetyl CoA Assay Kit ab87546 is a highly sensitive assay for quantifying Acetyl CoA level in  biological samples.


    In the Acetyl CoA assay protocol, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with a probe to generate fluorescence (Ex=535/Em=587 nm).


    The assay can detect 10 to 1000 pmol of Acetyl CoA (with detection limit ~0.4 µM).


    Acetyl CoA assay protocol summary:
    - add samples and standards to wells
    - add CoA quencher to wells to remove background from free CoA and succ-CoA and incubate at room temp for 5 min
    - add quencher remover and incubate at room temp for 5 min
    - add reaction mix and incubate for 10 min at 37ºC
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K317 PicoProbe™Acetyl-CoA Fluorometric Assay Kit. K317-100 is the same size as the 100 test size of ab87546.

    Acetyl CoA is a central molecule of metabolism. It carries acetate, used in the build-up and breakdown of larger molecules.

     

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests 2000 tests
    Acetyl CoA Assay Buffer WM 1 x 25ml 20 x 25ml
    Acetyl CoA Enzyme Mix Purple 1 x 500µl 20 x 500µl
    Acetyl CoA Standard (1 µmol) (Lyophilised) Yellow 1 vial 20 vials
    Acetyl CoA Substrate Mix (Lyophilised) Red 1 vial 20 vials
    CoA Quencher Orange 1 x 1ml 20 x 1ml
    Conversion Enzyme Green 1 x 100µl 20 x 100µl
    PicoProbe (in DMSO) Blue 1 x 200µl 20 x 200µl
    Quench Remover (Lyophilised) Clear 1 vial 20 vials
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    • Fatty acid oxidation
  • Relevance

    Acetyl CoA (AcCoA) is the metabolic intermediate which transfers carbon atoms to the TCA cycle for energy production and is the link between fat and carbohydrate metabolism. When fatty acid levels are high, AcCoA levels rise beyond the energy requirements of the organism and the excess is converted to the ketone bodies acetone and ß-hydroxybutyrate. AcCoA also contributes acetate to histone acetylation and isoprenoid synthesis.
  • Alternative names

    • AcCoA

Associated products

  • Related Products

    • Acetylcholinesterase Assay Kit (Colorimetric) (ab138871)
    • Acetylcholinesterase Assay Kit (Fluorometric - Green) (ab138872)
    • Acetylcholinesterase Assay Kit (Fluorometric -Red) (ab138873)

Images

  • Functional Studies - PicoProbe™ Acetyl CoA Assay Kit (ab87546)
    Functional Studies - PicoProbe™ Acetyl CoA Assay Kit (ab87546)Image from Zhang Y et al., PLoS One. 2016;11(3):e0150454. Fig 5(A).; doi: 10.1371/journal.pone.0150454. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Induction of acetyl-CoA in mitochondria by palmitate. Mitochondria were isolated from cells after palmitate treatment (300 μM) at 1, 4 and 16 h, and then used in the concentration assay of acetyl-CoA. 

    The acetyl-CoA content was determined immediately after isolation of the mitochondria using ab87546. Briefly, acetyl-CoA standard curve was made in the range of 0–100 pM and the correlation coefficient was 0.990 or higher. Protein was removed in the sample using the perchloric acid protocol and the supernatant was neutralized with 3 M KHCO3. The CoASH Quencher and Quencher remover were added into the sample to correct the background generated by free CoASH and succ-CoA. The sample was then diluted with the reaction mix, and the fluorescence signal was measured at Ex/Em = 535/589 nm with Spectra max Gemini XPS (Molecular Devices, Sunnyvale, CA). The relative acetyl-CoA concentration was normalized with the mitochondrial protein.

  • Functional Studies - PicoProbe™ Acetyl CoA Assay Kit (ab87546)
    Functional Studies - PicoProbe™ Acetyl CoA Assay Kit (ab87546)
    Examples of standard curves generated following the ab87546 kit protocol.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (48)

Publishing research using ab87546? Please let us know so that we can cite the reference in this datasheet.

ab87546 has been referenced in 48 publications.

  • Chen YY  et al. MCCC2 promotes HCC development by supporting leucine oncogenic function. Cancer Cell Int 21:22 (2021). PubMed: 33407468
  • Walker MA  et al. Acetylation of muscle creatine kinase negatively impacts high-energy phosphotransfer in heart failure. JCI Insight 6:N/A (2021). PubMed: 33554956
  • Möller J  et al. Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cells. Eng Life Sci 21:100-114 (2021). PubMed: 33716610
  • Rigby MJ  et al. Endoplasmic reticulum acetyltransferases Atase1 and Atase2 differentially regulate reticulophagy, macroautophagy and cellular acetyl-CoA metabolism. Commun Biol 4:454 (2021). PubMed: 33846551
  • Chen CL  et al. Arginine is an epigenetic regulator targeting TEAD4 to modulate OXPHOS in prostate cancer cells. Nat Commun 12:2398 (2021). PubMed: 33893278
View all Publications for this product

Customer reviews and Q&As

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1-10 of 19 Abreviews or Q&A

High background values in the fission yeast

Poor Average 3/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Protocol:
Two methods of sample preparation were tested.
1. Cell lysis by bead beating in acetyl-CoA assay buffer, deproteinization by 10 kDa spin column
2. Cell lysis by bead beating in TCA which also serves to denature proteins, neutralization by Na2CO3 to pH ~7
Extracts were prepared from fresh yeast biomass of S. pombe cells grown to exponential phase and processed as quickly as possible due to acetyl-CoA instability. Variable sample volumes in the assay - 5, 10 or 50 ul (samples diluted to 50 ul by assay buffer) were used. The standards and samples were measured in the 0-100 pmol range.

Figure legend:
A. Standard curve in the 0-100 pmol range. B. Fluorescence values of samples prepared from two yeast strains (A and B) using the 10kDa spin column. The acquired values of background samples (without conversion enzyme) are higher than those of samples. C. Fluorescence values of samples prepared from two yeast strains (A and B) using TCA precipitation. The acquired values of background samples is lower than those of samples only when 10 ul extract were used. Moreover, high volume (50 ul) of sample seems inhibitory for the assay.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Jarmila Tvarůžková

Verified customer

Submitted Feb 08 2018

Question

It is specified on page 9 to deproteinize the cell lysates but the following paragraph does not seem to concern only the lysates originating from tissues and not from cell culture.

Is deproteinization also necessary for cells in culture? if so, what are the proportions of perchloric acid per cell quantity?

Read More

Abcam community

Verified customer

Asked on Feb 08 2016

Answer

1.We recommend using ~2-5 X 10 ^ 6 cells for each manipulation.
2. Deproteinization is recommended for cells in culture. I have attached the protocol that the lab recommends for this step.

Read More

Sam Washer

Abcam Scientific Support

Answered on Feb 08 2016

Question

I have a question about the usage of this acetyl CoA kit for CoA measurements as well. Can this kit be used to measure CoA alone in the sample by eliminating the quencher and the conversion enzyme? If yes then do you provide a standard for CoA and what is the range of detection. Would it be similar to acetyl CoA standard curve?

Read More

Abcam community

Verified customer

Asked on Apr 17 2015

Answer

In principle, eliminating the quenching and conversion step will allow you to measure CoA alone. However, the kit was not produced nor validated for this purpose, so we cannot guarantee that it will work, nor do we have a CoA standard that has been validated with this method to provide with the kit.



Alternatively, I would recommend one of our CoA detection kits listed below so you don't have to optimize and piece together the assay yourself.



Colorimetric or Fluorometric detection (detection range; 2.5 µM - 250 µM)

https://www.abcam.com/coenzyme-a-detection-kit-ab102504.html



Fluorometric detection (detection range: 40 nM - 10µM)

https://www.abcam.com/coenzyme-a-detection-kit-fluorometric-green-ab138889.html

Read More

Kevin Hanson

Abcam Scientific Support

Answered on Apr 17 2015

Question

Il est précisé en page 9 de déprotéiniser les lysats cellulaires mais le paragraphe suivant ne semble concerner uniquement les lysats provenant de tissus et non de culture cellulaire.

La déprotéinisation est-elle aussi nécessaire pour des cellules en culture? si oui, quelle sont les proportions d'acide perchlorique par quantité de cellule?

Read More

Abcam community

Verified customer

Asked on Mar 18 2013

Answer



J’ai contacté le laboratoire concernant le kit ab87546 :

1.Nous recommandons d’utiliser ˜2-5 X 10^6 cellules pour chaque manip.

2. Il est recommandé de faire une déprotéinisation pour des cellules en culture. J’ai attaché le protocole que le laboratoire recommande pour cette étape.

3. Comme expliqué au téléphone, quand vous ajoutez le « CoA conversion » à vos échantillons, vous devez ajouter le mélange qui correspond à la gamme standard que vous utilisez. Par exemple, si vous pensez que vos échantillons sont faibles en acetyl CoA, utilisez la gamme et le mélange « CoA conversion » de 0-100 pmol.

4. Effectivement, il doit y avoir eu une inversion des tubes « Acetyl CoA Standard (1 µmol) (Lyophilised) » et « Acetyl CoA Enzyme Mix » et notre stock a maintenant été vérifié.

Read More

Abcam Scientific Support

Answered on Mar 18 2013

Question

For sample preparation from cultured cells, is it absolutely necessary to follow the specific cell lysis buffer provided in your protocol?
Second, for the deproteinization step, you recommend the use of perchloric acid method, could this be substituted by using a 10kD MW cut-off spin column?

Read More

Abcam community

Verified customer

Asked on Jan 23 2013

Answer

1. Yes it is very crucial to do the sample prep exactly as recommended as Acetyl CoA is very sensitive to degradation and you risk the chance of inefficient results if you make changes to the protocol.
2. The deproteinization approach, though not as sensitive as the sample prep, would still benefit from using PCA rather than the 10 kDa spin filters. But you can use the spin filters if you do not have the option of using PCA.

Read More

Abcam Scientific Support

Answered on Jan 23 2013

Question

  Inquiry: Hi, I am interested in using the PicoProbe based assay kits. Before placing an order I would like to know if picoprobe is specific to NADH or will it give a positive response to other co-factors as well.

Read More

Abcam community

Verified customer

Asked on Jun 08 2012

Answer

Thank you for contacting us.

I have had the opportunity to discuss the specificity of this kit with the lab as you have requested. I have found that in this particular assay the probe is specific to just the NADH.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Abcam Scientific Support

Answered on Jun 08 2012

Question

I am interested in this acetyl-CoA kit but I need more detail information about how this kit works to measure Acetyl-CoA. i) Does Acetyl CoA enzyme take the responsibility of the reaction from acetyl-CoA to acetate+CoA? ii) What is substrate-mix for? iii) What is conversion enzyme for? Thanks for your information.

Read More

Abcam community

Verified customer

Asked on May 30 2012

Answer

Thank you for your interest in our acetyl CoA assay kit, ab87546.

The assay measures relative amounts of acetyl CoA in samples indirectly, compared to a standard included with the kit, by converting the acetyl CoA to CoA, and then using the resulting CoA to reduce NAD to NADH, which in turn reacts with the probe to generate a fluorescent signal .

The conversion enzyme is what converts the acetyl CoA to CoA. The substrate mix is what the CoA acts upon, to generate the NADH. The components of the substrate mix and the identity of the conversion enzyme are considered proprietary information.

I hope this helps. Please let me now if you have any other questions.

Read More

Abcam Scientific Support

Answered on May 30 2012

Question

Once sample is prepared, can we store it and at what temperature?

Read More

Abcam community

Verified customer

Asked on Feb 10 2016

Answer

The sample can be stored at -80 degree centigrade once prepared. However, we recommend to use the sample immediately after preparation for maximum efficiency.

Read More

Sam Washer

Abcam Scientific Support

Answered on Feb 10 2016

Question

I would like to know if your kit PicoProbe Acetyl CoA Assay Kit (ab87546) is suitable for mouse frozen tissues.

Read More

Abcam community

Verified customer

Asked on Nov 20 2014

Answer

I can confirm that kit PicoProbe Acetyl CoA Assay Kit (ab87546) can be used with frozen mouse tissues. Please see the protocol booklet for more information regarding sample preparation.

Read More

Abcam Scientific Support

Answered on Nov 20 2014

Question

Is this PicoProbe Acetyle CoA Assay kit, compatible with the mitochondrial sample isolated from cultured cells?

What is the recommended amount of sample added for each reaction?

Read More

Abcam community

Verified customer

Asked on Aug 29 2013

Answer

1. Mitochonrial lysate can be assayed by ab87546.

2. For the amount sample to be added into each sample well, we recommended to perform a pilot experiment with a range of dilutions of the sample to determine the optimal concentration/dilution of sample which gives the best results in the linear range of the standard curve. Please note that the probe in this kit is a very sensitive probe (minimum detection level is 10 pmol of Acetyl CoA). You may start with 1 ul sample to 10ul sample per well.

Read More

Stanley Leung

Abcam Scientific Support

Answered on Aug 29 2013

1-10 of 19 Abreviews or Q&A

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